Sjogren's syndrome is a chronic autoimmune disease, which results in decreased secretion from salivary and lacrimal glands. The broad, long-term objectives of this proposal are to define pathogenic mechanism(s) in Sjogren's syndrome, with a view to identifying relevant pathways for therapeutic intervention in this disease. Recent studies have implicated abnormal cholinergic signaling and the cytotoxic lymphocyte granule pathway as areas of potential importance in this regard. This proposal will focus on these pathways in Sjogren's syndrome, by defining the mechanisms by which granzyme B (GrB) induces salivary gland epithelial cell damage and dysfunction and autoantibody production. The specific aims of the proposal are to (1) Define the mechanisms and functional effects of type 3 muscarinic acetylcholine receptor (M3R) cleavage by GrB. This will be done by elucidating GrB-induced cleavage of M3R that occurs in intact cells during granule-induced cytotoxicity, and addressing the effects of GrB cleavage on M3R ligand binding and signal transduction; (2) Identify the predominant mechanisms of salivary epithelial cell damage and dysfunction in vivo in patients with Sjogren's syndrome. Specific evidence of GrB- generated fragments of Sjogren's syndrome autoantigens in affected tissue from patients with Sjogren's syndrome will be sought, using novel antibodies that are highly specific for neo- epitopes generated by GrB-mediated cleavage. The frequency of antibodies to the M3R in patients with primary or secondary Sjogren's syndrome will be quantitated; and (3) Establish the role of GrB in the development of salivary gland dysfunction and damage in animal models in vivo. This will be accomplished by studying the development of a Sjogren's syndrome-like phenotype (inflammatory infiltrates, autoantibodies and secretory dysfunction) in wild-type and GrB-deficient NOD mice, and addressing whether immunization with granule-killed primary salivary epithelium results in an autoantibody response against salivary gland epithelial cell GrB substrates, including M3R, with functional consequences. These studies will yield an enhanced understanding of the mechanisms whereby the cytotoxic lymphocyte granule pathway participates in the pathogenesis of Sjogren's syndrome.